Overexpression of AHL9 accelerates leaf senescence in Arabidopsis thaliana

Background Leaf senescence, the final stage of leaf growth and development, is regulated by numerous internal factors and environmental cues. Ethylene is one of the key senescence related hormones, but the underlying molecular mechanism of ethylene-induced leaf senescence remains poorly understood. Results In this study, we identified one AT-hook like (AHL) protein, AHL9, as a positive regulator of leaf senescence in Arabidopsis thaliana. Overexpression of AHL9 significantly accelerates age-related leaf senescence and promotes dark-induced leaf chlorosis. The early senescence phenotype observed in AHL9 overexpressing lines is inhibited by the ethylene biosynthesis inhibitor aminooxyacetic acid suggesting the involvement of ethylene in the AHL9-associated senescence. RNA-seq and quantitative reverse transcription PCR (qRT-PCR) data identified numerous senescence-associated genes differentially expressed in leaves of AHL9 overexpressing transgenic plants. Conclusions Our investigation demonstrates that AHL9 functions in accelerating the leaf senescence process via ethylene synthesis or signalling. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-022-03622-9.


Background
Senescence is an intricate and highly orchestrated process in the plant's life cycle. A range of biological events occur at the physiological, biochemical, and molecular levels during this period, including chloroplast degradation, hydrolization of macromolecules, reduction of cytoplasmic volume, and decrease in cellular metabolic activity. The most noticeable feature in leaf senescence is the rapid degradation of chlorophyll during chloroplast disassembly, which leads to the yellowing of leaves [1].
During senescence, leaf nutrients are remobilized and relocated from the dying leaves to seeds or other storage tissues, thereby contributing to the fitness and survival of plant [2][3][4]. Although senescence is an active process to relocate nutrients from old tissues, precocious senescence will shorten the growth stage of crops and result in reduced yield and crop quality [2,5,6].
Senescence involves massive transcriptional changes, and a large number of senescence-associated genes (SAGs) have been identified. The expression of SAGs is regulated by senescence-related transcription factors TFs such as NACs (NAM [No Apical Meristem], ATAF2 [Arabidopsis Transcription Activation Factor2] and CUC2 [Cup-shaped Cotyledon2]), MYBs, bZIPs (basic region/leucine zipper motifs) and WRKYs TFs [7][8][9][10]. Incubation of detached leaves in the dark is highly effective for induction of SAGs, leaf yellowing and chlorophyll loss. Therefore, it has been widely used as a model system for the study of leaf senescence [11].
Although leaf senescence occurs in an age-dependent manner, the process is also greatly affected by multiple endogenous and environmental signals coordinating the life span of leaves to optimize plant fitness. Endogenous signals such as plant hormones play vital roles in the senescence process via complex interconnecting pathways. Positive regulators include abscisic acid, ethylene, jasmonic acid, salicylic acid, brassinosteroid and strigolactone, in contrast, cytokinin, gibberellic acid and auxin suppress senescence [12,13]. Ethylene has long been established as a key hormone regulating the timing and progression rate of leaf senescence [14,15]. In vitro application of ethylene induces premature leaf senescence, while application of inhibitors of ethylene biosynthesis or action can delay leaf senescence symptoms [16,17].
Transcriptional analysis has shown that the expression levels of a number of genes encoding ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and ACC oxidase (ACO), and signaling components increase in senescence leaves [16], indicating ethylene signaling is involved in the regulation of senescence leaves. This is further supported by the extended leaf longevity of ethylene-insensitive mutants that are defective in ethylene signaling transduction, such as etr1 (ethylene resistant 1), ein2 (ethylene insensitive 2) and ein3 [18][19][20]. To date, the regulation of ethylene seems to be achieved by the EIN2-EIN3-miR164-NAC2 signalling cascade. EIN2 regulates miR164 expression, as well as its downstream target gene ORESARA1 (ORE1) ORE1/NAC2 [21]. EIN3 acts at the downstream of EIN2 to promote chlorophyll degradation by affecting chlorophyll catabolic genes [22]. Although the ethylene biosynthetic pathway and downstream key elements involved in ethylene signal transduction have been extensively studied through genetic approaches, the transcriptional network leading to leaf senescence remains largely unknown.
AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) proteins are transcription factors featured with two conserved structural units: a plant and prokaryote conserved (PPC) domain, involved in protein-protein interactions, and one or two DNA-binding AT-hook motif(s) [23][24][25][26][27]. The AT-hook motif contains a conserved palindromic core sequence, Arg-Gly-Arg, capable to bind to the minor groove of AT-rich B-form chromosomal DNA, thus changing its architecture and controlling the expression of corresponding genes [25,[28][29][30]. AHL family proteins have been proposed to regulate plant growth and development. AHL22 acts as a chromatin remodeling factor that regulates FT (FLOW-ERING LOCUS T) expression to promote flowering [31]. Several AHLs are involved in hormonal homeostasis and response, especially gibberellins, cytokinins and jasmonic acid [28,32,33]. ESC/AHL27 (ESCAROLA) and SOB3/AHL29 (SUPPRESSOR OF PHYTOCHROME B-4 #3) act redundantly to repress hypocotyl elongation in response to light [24,34]. Overexpression of AHL27 in Arabidopsis delays senescence and increases post-harvest storage life, but the molecular mechanism is unclear [29]. Recently, it has been reported that SOB3/AHL29 repress petiole growth by antagonizing PIF-mediated transcriptional activation of genes associated with growth and hormonal pathways [35].
Although the fact that one member of the AHL clade A is involved in leaf senescence, the detailed mechanism by which AHL transcription factors regulate leaf senescence still remain largely unknown. Here, we identified AHL9 as a positive regulator of leaf senescence in Arabidopsis. The overexpression of AHL9 causes a severely early senescence phenotype. Moreover, we showed in the current study that AHL9 not only promotes dark-induced but also ethylene-induced leaf chlorosis. Our RNA-seq analysis showed that the expression of multiple SAGs is altered in AHL9 overexpressing lines. Our results provide new insights into the molecular mechanism of leaf senescence highlighting AHL9 as a prominent regulatory component in dark-induced and ethylene-induced leaf senescence.

Results
Overexpression of AHL9 results in premature leaf senescence AHL family proteins include clade A and clade B subfamilies [24,26]. To further explore the functions of AHL clade B subfamily, a phylogenetic tree was generated and analysed ( Fig. S1). AHL9 was chosen to study its biological functions. Bioinformatics analysis demonstrates that AHL9 genome fragments contain 5 exons and 4 introns, the AHL9 protein has two AT-hook motifs and one DUF296 (plant and prokaryote conserved (PPC)/domain of unknown function #296 domain) (Fig. S2). AHL11 is the closest homolog of AHL9 in Arabidopsis genome, which exhibits 62.2% sequence identity with AHL9 based on full-length alignment (Fig. S3). Through the quantitative reverse transcription PCR (qRT-PCR) assay, various expression abundancy of AHL9 was detected. However, a higher expression in the aging tissues (the fourth and fifth rosette leaves) than in proliferative tissue (the siliques) was observed, indicating AHL9 may have the role during the senescence process (Fig. S4). To test the biological function of AHL9, 35S::AHL9 transgenic Arabidopsis lines were generated. Two independent transgenic lines (35S::AHL9-OE10 and OE11) were further analysed. Compared with WT, both transgenic lines displayed premature leaf senescence at 32 d (Fig. 1A). In addition to precocious leaf senescence, the rosette leaves of both AHL9 transgenic lines became elongated and narrow (Fig. 1A). To gain a better view of the function of AHL9 in leaf senescence, the rosette leaves of transgenic lines were compared with their corresponding WT at the same stage. As shown in Fig. 1B, when the first five leaves of AHL9-OE10 and AHL9-OE11 had already turned yellow, only the first three leaves became yellow in WT. In contrast with an apparent premature senescence of AHL9 transgenic lines, the AHL9 T-DNA insertion mutant was almost like the WT (Fig. S5). Phylogenetic analysis showed that AHL9 hold the similarity with AHL11, AHL5 and AHL12, indicating the possible functional and arranged according to their age. Rosette leaves were numbered from bottom to top with the first leaf being the oldest. Scale bar = 1 cm. C Representative images of the fourth and fifth rosette leaves detached from WT, AHL9-OE10 and AHL9-OE11 plants of the different ages. D Chlorophyll contents of the leaves shown in (C). *P < 0.05, **P < 0.01, Data indicate means ± SD, n = 3. Statistical analyses were performed using one-way ANOVA. Means with different letters above the bars indicate statistically significant results (P < 0.05). The experiment was conducted three times with similar results redundancy. As AHL11 exhibited the highest similarity with AHL9, we generated the double mutants that lack of both AHL9 and AHL11. All plants (WT, ahl11, ahl9 ahl11-1 and ahl9 ahl11-2) exhibited the similar rate in leaf senescence, implying the higher-order redundancy among the proteins belonging to AHLs family. (Fig. S6).
To further analyse the development of age-related senescence in WT and AHL9 overexpressing plants, the fourth and fifth rosette leaves detached from WT and 35S::AHL9 transgenic lines were used for further analysis in the different stages. At 20 and 26 d after germination, there was no significant difference in leaf colour between transgenic lines and WT. However, at 32 d after sowing, the fourth and fifth rosette leaves of 35S::AHL9 transgenic lines became yellow, while the same stage leaves of WT were still green. The yellowing of these leaves progressed rapidly in the AHL9-OEs transgenic lines, and the majority of them become completely yellow at 38 d after germination, at this stage, WT plants only started to show signs of leaf senescence with only the tip of the leaf turning yellow (Fig. 1C). This is consistent with previous reports showing that senescence symptoms usually start from the tip and outer edge of a rosette leaf at a given age [36]. Chlorophyll quantification assays confirmed the visual phenotypic observations showing significantly lower chlorophyll content in the fourth and fifth rosette leaves in AHL9 overexpressing transgenic lines in 32 d and 38 d-old plants, compared with WT plants (Fig. 1D).

AHL9 regulates dark-and ethylene-induced leaf senescence
Incubation of detached leaves in darkness is often used as an effective method to stimulate synchronous senescence [37]. To further probe the potential roles of AHL9 in leaf senescence, we examined dark-induced leaf senescence in three-week-old WT and 35S::AHL9 transgenic plants. Rosette leaves of the same age detached from 3-week-old WT and 35S::AHL9 plants were similar in colour before dark treatment; however, after 3 days darkness, 35S::AHL9 leaves exhibited stronger leaf chlorosis than WT (Fig. 2A). The phenotypic observations were confirmed by chlorophyll assays showing a more pronounced decrease in chlorophyll levels in the AHL9-OE leaves compared to WT (Fig. 2B), suggesting that darkinduced leaf senescence is promoted by the overexpression of AHL9.
Phytohormones play critical roles in leaf senescence, therefore we queried whether the overexpression of AHL9 altered the ethylene-and ABA-induced senescence. For this purpose, we first examined the darknessinduced senescence of detached leaves upon treatment with either the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or ACC plus aminooxyacetic acid (AOA), an ethylene biosynthesis inhibitor, respectively. As expected, pre-treatment with ACC increased senescence in WT and 35S::AHL9 leaves (Fig. 2C, D). In contrast, the enhanced senescence observed in 35S::AHL9 leaves was suppressed by treatment with ACC and AOA (Fig. 2C, D), suggesting that the role of AHL9 in senescence is dependent on ethylene. Unlike ethylene, ABA treatment did not significantly increase the difference of chlorophyll content between WT and AHL9 overexpression transgenic lines (Fig. S7). These results indicate the involvement of AHL9 in ethylene-induced leaf senescence.

AHL9 is a nuclear localized AT-hook protein
To establish the AHL9 subcellular localization, an AHL9-GFP fusion construct or GFP (control) were transiently expressed in Arabidopsis mesophyll protoplasts under the control of the cauliflower mosaic virus 35S promoter. The nuclear marker protein H2B-mCherry was co-expressed to visualize nuclei [38]. In the GFP controls, green fluorescence was observed throughout the protoplasts (Fig. 3). Green fluorescence in AHL9-GFP transfected protoplasts was restricted to the nuclei and overlaped with the yellow fluorescence of the nuclear marker, indicating that AHL9-GFP is located in the nucleus (Fig. 3).

Identification of differentially expressed genes in AHL9 overexpression lines
In order to investigate the possible roles of AHL9 in leaf senescence, we performed genome-wide expression profiling of AHL9-OEs and WT plants under normal growth conditions. Leaves from 30-d old WT and both AHL9-OEs transgenic lines were used for RNAseq experiments. In total 20 million uniquely mapped reads per sample with high reproducibility among all three biological replicates were generated (Table S1). Analysis of AHL9-OE10 vs WT identified 953 genes with statistically significant differences in gene expression (log 2 (fold_change) > 1, q-values < 0.01), including 342 up-regulated and 611 down-regulated genes (Fig. 4A-B and Table S2). In the case of AHL9-OE11, 1451 genes showed statistically significant differences with WT, with 556 up-regulated and 895 down-regulated genes. When both datasets are put together, a common set of 665 differentially expressed genes (DEGs) were identified in AHL9-OE10 and AHL9-OE11 vs WT (Fig. 4C). Interestingly, some of the genes with altered expression belong to NAC, WRKY, IAA, and AP2 transcription factors, ACC oxidase, and the ETHYLENE RESPONSE SENSOR (Fig. 4D). Principal component analysis (PCA) and correlation analysis of all datasets indicated strong RNA-seq reliability (Fig. S8-S9). qRT-PCR analysis of 10 randomly . The data were analyzed using one-way ANOVA analysis. Means with different letters above the bars indicate statistically significant results (P < 0.05). Data indicate means ± SD, n = 3. The experiment was conducted three times with similar results. C Phenotype of detached leaves from 3-week-old WT and two independent AHL9 overexpressing lines ACC or ACC + AOA in the dark. Detached leaves were treated with MES buffer, 100 µM ACC or 100 µM ACC + 500 µM AOA for 3 d under the dark conditions. D Chlorophyll contents in leaves from (C). The data were analyzed using one-way ANOVA analysis. Means with different letters above the bars indicate statistically significant results (P < 0.05). Data indicate means ± SD, n = 3. The experiment was conducted three times with similar results Fig. 3 AHL9 is localized exclusively in the nucleus. Arabidopsis protoplasts were co-transfected with expression cassettes containing either 35S::GFP and 35S::H2B-mCherry or 35S::AHL9-GFP and 35S::H2B-mCherry. GFP signals were detected using a laser confocal scanning microscopy. H2B-mCherry was used as a nuclear marker. From left to right are green fluorescence signal, nuclear marker, chlorophyll red auto fluorescence, bright-field and merged images, respectively. Scale bars = 10 μm chosen DEGs are consistent with the RNA-seq results (Fig. 4E, Table S3). Gene Ontology analysis highlighted the expression changes of genes involved in response to stimuli, hormone response, biological process and biological regulation (Fig. 5). Given that gene regulatory networks composed of interactions between TFs (transcription factors) and their targets have been implicated in controlling leaf senescence, we conducted GO enrichment analysis for these TFs. Indeed, we found most of these TFs were significantly enriched in GO terms that may associate with biological process, such as the RNA biosynthetic or metabolic process, cellular macromolecule biosynthetic process and nitrogen compound metabolic process (Fig. S10). These results provided molecular evidence supporting premature leaf senescence in AHL9 overexpression lines.

Discussion
In recent years, our knowledge about the molecular mechanisms triggering leaf senescence has expanded significantly. Transcriptomic analysis of leaf senescence revealed the expression changes of thousands of SAGs, however, only a small portion of them have been functionally characterized [8]. Besides, factors on the top of the regulation module regulating diverse SAGs and/or other functional genes have also been identified through either loss-of-function and/or gain-of-function studies in model plants such as Arabidopsis and rice [2,23]. Among these regulators, transcription factors are interesting candidates as they can influence the expression of multiple genes during the senescence process.
The study of AHL family genes revealed different roles in plant growth and development, such as hypocotyl elongation, flower development, gibberellin biosynthesis and leaf longevity [25,28,31,34]. Although knowledge about the functional role of AT-hook motif proteins is still very limited in plants. Notably, Ectopic ORE7/ ESC delays leaf senescence, which probably up-regulate genes that suppress senescence and down-regulate genes that enable the progression of the senescence process through modification of chromatin architecture [29]. In this study, we identify and characterize an AHL protein, AHL9, involved in leaf senescence. Overexpression of AHL9 results in early senescence in Arabidopsis (Fig. 1). Transgenic plants overexpressing AHL9 also exhibited accelerated dark-induced leaf senescence ( Fig. 2A). In contrast, the lack of senescence-related phenotype in the ahl9 mutant may be due to the existence of functionally redundant genes, since 4 paralogs were identified in Arabidopsis, including AHL5, AHL12, AHL9, and AHL11 [26]. The lack of phenotype in the ahl9 mutant could also be explained by functional compensation from other senescence-associated pathways, since leaf senescence is the integrated result of various pathways that incorporate numerous endogenous factors and environmental signals [8,23]. Taken together, these results suggest that AHL9 behaves like an early senescence-activator that influences both age and dark-induced leaf senescence.
Although ethylene has been known for many decades to be a senescence-inducing plant hormone, the molecular mechanism underlying ethylene-mediated senescence remains largely unknown. Comparative transcriptome analyses have revealed a number of ethylene biosynthesis and signalling genes, with elevated transcript levels in senescing leaves, supporting the idea that sensitivity of a leaf to ethylene might account for the age-dependent leaf senescence [16,18]. Here, we show that AHL9 is involved in the control of leaf senescence through ethylene synthesis or signalling since pre-treatment with the ethylene biosynthesis inhibitor AOA, can effectively repress the increased senescence observed in AHL9 overexpressing transgenic lines. Consistent with this data, some ethylene synthesis and signalling genes are induced in the RNA-seq data, although the genes with altered expression may not be the direct targets of AHL9 (Fig. 4D). Intriguingly, we also find some of these TFs that show DEGs are significantly enriched in GO terms that may associated with stress response such as response to abiotic stimulus, ethylene-activated signaling pathway and hormone-mediated signaling pathway. Ethylene is sensed by a five-member family of ethylene receptors on the endoplasmic reticulum [39], then this binding inactivates a Raf-like Ser/Thr kinase, CTR1 (CONSTITUTIVE TRIPLE RESPONSE1), thereby releases the C-terminal end of the positive regulator, EIN2, to the nucleus and stabilizes EIN3 and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), which in turn, activate the expression of ethylene target genes to promote premature senescence in leaves [40][41][42][43]. Identifying specific target genes and interaction proteins of AHL9 in vivo will be the next step required to gain a more in-depth knowledge of its role in regulating leaf senescence.

Conclusions
The current study demonstrates that one AT-hook protein, AHL9, may regulate leaf senescence via the finetuning of ethylene biosynthesis or signalling.

Plant materials and growth conditions
The Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study [44]. The transfer DNA (T-DNA) insertional mutant ahl9 (GK_735D06) was obtained from the Nottingham Arabidopsis Stock Centre (NASC). Seeds were surface sterilized in 10% (v/v) sodium hypochlorite for 10 min, washed 3 times with sterilized water, and then grown on Murashige and Skoog medium plus 3% sucrose and 0.6% agar (pH 5.8) after 2 d vernalization in darkness at 4℃. The 7-d-old seedlings were transferred into soil and were grown at 22℃ in a 16-h-light/8-h-dark cycle for additional experiments and seed production. Yusen Zhou and Jing Chen undertook the formal identification of the plant materials (T-DNA mutants, CRISPR-Cas9 mutants and transgenic plants) used in this study.

Plasmid construction and plant transformation
AHL9 cDNAs were obtained by RT-PCR of RNA isolated from Col-0 seedlings, the full length of AHL9 CDS was amplified by PCR method using primers. According to the instructions of the invitrogen gateway kit (kit No.11789 (BP Clonase); No.117910 (LR Clonase)), the PCR products were cloned into pDNOR221 vector using the Gateway ™ BP Clonase ™ II Enzyme mix. Subsequently, AHL9 CDS was sub-cloned into the pDEST Gateway binary vector pGWB405 between the 35S promoter and GFP gene to produce the 35S::AHL9-GFP construct. The constructed plasmids were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into plants using the floral dip method [45]. Transgenic plants in the T 2 generation with T-DNA insertion at a single locus were selected by kanamycin resistance, and T 3 homozygotes were used for all analyses. These primers were listed in Table S3.

Dark-induced senescence assay
For the dark-induced senescence assay, the fourth and fifth rosette leaves were carefully detached from 3-weekold soil-grown Arabidopsis. Detached rosette leaves were incubated on MES buffer (0.5 × MS, 3 mM MES, pH 5.8) in complete darkness for 3 d and sampled for analyzing leaf senescence and chlorophyll content.

Hormone induced leaf senescence assay
The fourth and fifth leaf of 3-week-old plants were detached and floated on 3 mL of MES buffer (0.5 × MS, 3 mM MES, pH 5.8) supplemented with or without ABA (10 μM, 50 μM, 100 μM, and 200 μM), 1-aminocyclopropane-1-carboxylic acid (ACC, 100 μM) and 100 μM ACC plus ethylene biosynthesis inhibitor (AOA, 500 μM). The petri dishes were sealed with parafilm tape and wrapped with double-layer aluminum foil, then the petri dishes were put in a black box to avoid light. All hormone treatments were performed at 22℃ under the dark conditions. Three biological replicates were performed.

Determination of chlorophyll content
Chlorophyll was measured according to the method described by Li et al. [47]. Briefly, Arabidopsis seedlings were weighed (W), placed into Eppendorf tubes with acetone (95%, 1 mL, V) and kept overnight under dark conditions. Samples were then centrifuged at 13,000 g for 10 min and absorbance values at 665 nm and 649 nm were obtained from the supernatant. Chlorophyll content (including chlorophyll a and b) was calculated according to the following formula: chlorophyll a = 13.95A665 -6.88A649; chlorophyll b = 24.96A645 -7.32A665; and total chlorophyll = (chlorophyll a + chlorophyll b) × V/W. At least three independent samples were examined, all of which produced the typical results reported in this article.

qRT-PCR analysis
Total RNA was isolated from the seedlings with Trizol reagent and DNA was digested by RNase-free DNase I. Two µg of total RNA was used for reverse transcription with M-MLV reverse transcriptase according to the supplier's instructions (Promega). qRT-PCR analyses were performed with Roche Light Cycler 480 real-time PCR system using the SYBR Green Master Mix (Vazyme Biotech Co., Ltd.) and specific primers for PCR amplification. UBQ10 was used as an internal control for data normalization. These primers were listed in Table S3.

RNA-seq analysis
The sixth, seventh and eighth rosette leaves were individually collected from 30-d-old WT, AHL9-OE10 and AHL9-OE11, pooled, and frozen in liquid nitrogen. The samples were stored at − 80℃ prior to RNA extraction. The RNA-seq analysis was performed at the Berry Genomics Corporation (Beijing) with three biological replicates. Briefly, The cDNA library were constructed and sequenced on HiSeq 2000 sequencing system. The clean data was produced by discarding the paired reads that one read's number of N base is more than 5 or has more than 30% bases with a low quality value below 15. The clean reads were aligned to the reference genome of Vigna angularis using Hisat2 [49]. Then samtools and HTSeq-count were used to count the reads number of each gene and gene's expression level was normallized as Fragments Per Kilobase Million (FPKM) [50]. DESeq2 was used to analyze differential gene expression, based on the negative binomial distribution, of the replicate samples between the treatment group and control group [51]. A threshold value of p-adjusted value (qvalue) < 0.01 (|log 2 foldchange|> 1) was used to obtain differentially expressed genes (DEGs). The gene ontology (GO) enrichment, with p-adjusted value cut-off 0.05, were performed using custom R scripts based on Bioconductor packages goseq and GO.db in order to classify DEGs into terms.

Phylogenetic tree
Phylogenetic tree was generated using MEGA-X software. The AHL clade B family from Arabidopsis thaliana, some orthologs of rice and maize protein sequences were downloaded from NCBI.

Statistical analysis
All experiments were repeated with at least three times.
The presented data were expressed as the means ± SD.